Variation in allelic expression is common in humans (Lo, H. S. et al., Genome Res 13:1855-62, 2003, Hoogendoorn, B. et al., Hum Mol Genet. 12:2249-54, 2003) and, underlies normal human variability and predisposition to human diseases (Yan, H. et al., Science 297:1143, 2002). Genomic imprinting represents the extreme end of this expression spectrum where only one allele is expressed in a parent-dependent manner. Normal genomic imprinting is regulated, in part, by methyl groups on cytosines within cytosine-guanine (CpG) dinucleotides, i.e., cytosine-deoxyribose phosphates adjacent to a guanine-deoxyribose phosphate. In principle, DNA methylation within regions differentially methylated between parental alleles (differentially methylated regions, DMRs) can predict normal imprinting or loss of imprinting.
As a proxy for parent-specific expression, current methodologies based on DNA methylation are either 1) labor-intensive (bisulfite genomic sequencing) (Frommer, M. et al., Proc Natl Acad Sci USA 89:1827-31, 1992) or; 2) do not assess parent-specific methylation levels but instead, average methylation levels (Dupont, J. M. et al., Anal Biochem 333 :119-27 (2004). Bisulfite genomic sequencing is a method that quantities methylation at a similar resolution and represents the gold standard in the field. However, bisulfite genomic sequencing requires the labor-intensive of subcloning PCR-amplified products into vectors prior to direct sequencing of multiple individual clones that each represent one parental strand from a single cell. Artifacts may result from strong assumptions that do not correlate with normal biological heterogeneity. For example, an average 50 percent methylation of a population of cells may represent either the assumed normal imprinting in the entire cellular population (zero methylation on one allele; 100 percent, the other) or variations of loss of imprinting (e.g. an allele with 75 percent methylation and another allele harboring 25 percent methylation).
Therefore, there is a pronounced need in the art for novel methods having substantial utility for assessing the degree of allele-specific methylation present in cells and tissues.